Method for Prongf Assay for in Vitro Diagnosis of Cancer in Particular Breast, Thyroid or Lung Cancer, and Therapeutic Use of Prongf

ABSTRACT

The present invention concerns a method for in vitro diagnosis of cancer, in particular breast, thyroid or lung cancer, which consists in determining the presence of ProNGF in a biological sample derived from a patient suspected of suffering from cancer, in particular breast, thyroid or lung cancer. Said method may be used for early diagnosis, screening, therapeutic follow-up, prognosis and for diagnosing relapses in cancer and in particular breast, thyroid or lung cancer. 
     The present invention also concerns the use of a ProNGF inhibitor for preparing a drug, said drug being in particular useful for blocking remote dissemination and cell invasion in patients suffering from cancer, in particular breast, thyroid or lung cancer. 
     The invention is applicable in the field of diagnosis and therapeutics.

The present invention relates to the cancerology field. Moreparticularly, the present invention relates to a method for diagnosingcancer, and more particularly breast, thyroid, lung or prostate cancer,in a human patient by in vitro determination of the presence of thenerve growth factor precursor (ProNGF) in a biological sample derivedfrom this patient, or in the patient's tumor in vivo, it being possiblefor said method to be used for early diagnosis, screening, therapeuticfollow-up and prognosis, and also for diagnosing relapses in cancer.Furthermore, due to the capacity of cancer cells, and more particularlybreast, thyroid, lung or prostate cancer cells, for producing ProNGF,the present invention also relates to therapy.

In women, breast cancer is the most common cause of mortality due tocancer in industrialized countries. It is estimated that the minimumsize of a tumor that can be detected by mammography is a few millimeters(mm). Breast cancers develop slowly. Nevertheless, at the time thissmall tumor is diagnosed, it has already been evolving for on average 8years. The etiology of breast cancer is not well defined. Familialpredispositions have been demonstrated. Age is the most important riskfactor. Thus, the risk increases by 0.5% per year of age in westerncountries. Other risk factors are known, such as the number ofpregnancies and the age at the first pregnancy, breast feeding, the ageat puberty and at menopause, oestrogenic treatments after the occurrenceof the menopause, stress and nutrition.

The test that is available and used in mass screening for breast canceris an imaging technique: mammography. By virtue of this technique,mortality due to breast cancers has greatly decreased (30% reduction inmortality), which underlines the importance of tumor screening in termsof public health. Nevertheless, the screening techniques suffer from acertain number of handicaps. Mammography requires high-performanceequipment and qualified personnel, which is expensive in the context ofmass screening. Thyroid cancer is a rare cancer. It representsapproximately 1% of cancers occurring in the general population inFrance. Its annual incidence is low, i.e. approximately 2.5 per 100 000individuals (Cancers: évaluation, traitement et surveillance [Cancers:assessment, treatment and monitoring]. J M Andrieu & P Colonna Ed.ESTEM, Paris 1997). The number of new cases of thyroid cancer in theUnited States in 2006 is estimated at 30 000, and the deaths at 1500(American Cancer Society: Cancer Facts and Figures 2006. Atlanta, Ga.:American Cancer Society, 2006).

Thyroid cancer usually develops in the form of a nodule located withinthe thyroid gland, whether it is of normal size or enlarged (goitre). Itis a rare cancer, more common in young individuals, for which theprognosis is good when the cancer is in its papillary form sincerecovery occurs in 90% of cases.

The prevalence of the thyroid nodule is variable according to thescreening means. It is more common in women, elderly individuals, andindividuals who live in an area where there is an iodine deficiency orwho have undergone irradiation of the cervical region during infancy,but these nodules are benign in more than 90% of cases. Youngindividuals are more exposed to development of the cancer due to agreater sensitivity of the thyroid to irradiation.

According to the international histological classification, adistinction can be made between four main histological types of thyroidcarcinoma:

-   -   papillary epitheliomas,    -   follicular epitheliomas,    -   medullary epitheliomas,    -   anaplastic (or undifferentiated) epitheliomas.

These tumors may be solitary or multifocal.

The papillary cancers are more common. They predominate in youngindividuals and represent approximately 80% of thyroid cancers.

The follicular cancers represent approximately 10% of thyroid cancersand are especially common around the age of about forty.

The papillary and follicular cancers represent the group ofradiosensitive differentiated thyroid cancers. They secretethyroglobulin.

The medullary cancers represent 5% of thyroid cancers and correspond toa tumor of the C or parafollicular cells derived from the neural crest.The C cells secrete calcitonin. The anaplastic or undifferentiatedcancers are rare (less than 5% of cases) and extremely serious.

In the presence of a malignant thyroid nodule, the basic treatment issurgery. If residual functional tissue remains, a dose of iodine 131 isadministered in an isolated chamber, 4 to 6 weeks after a totalthyroidectomy, in order to entirely sterilize it. Approximately fortypercent of thyroid cancer metastases fix iodine and can therefore betreated by this method.

After total thyroidectomy and sterilization of the thyroid with iodine131, thyroxine, a hormone which inhibits TSH secretion, is administered.This hormone therapy also makes it possible to ensure a satisfactorythyroid balance in functional terms. The discovery of new diagnostic andprognostic markers and of a targeted therapeutic agent could make itpossible to complete the therapeutic and diagnostic arsenal for thiscancer.

Lung cancer, which is responsible for more than 25 000 new cases eachyear in France, can be considered to be a major public health problem.It is the most common cancer in men and in fact represents the mostcommon cause of mortality due to cancer in men and the third most commoncause in women. The number of new cases of lung cancer (non small celland small cell combined) in the United States in 2006 is estimated at174 470, and deaths at 162 460 (American Cancer Society.: Cancer Factsand Figures 2006. Atlanta, Ga.: American Cancer Society, 2006).

Within the primary cancers, examination of the cancer cells(anatomopathological examination) makes it possible to draw adistinction between:

-   -   Epidermoid cancers (35-40%);    -   Adenocarcinomas (25-35%);    -   Large cell carcinomas (10-15%);    -   Small cell carcinomas (20-25%).

These four categories represent close to 95% of lung cancers. The firstthree are grouped together as “non small cell lung cancer” (NSCLC).

Small cell cancer progresses much more rapidly and is more likely tospread to other organs.

Carcinoid tumors and muco epidermoid tumors are more rare, and representthe remaining 1 to 2%.

These classifications can be summarized as “small cell lung cancer”(13%) or “non small cell lung cancer” (87%) with different therapeuticimplications. Efforts at early detection have not been shown to beeffective with the current tools (lung X-ray, analysis of sputum andfibroscopy do not improve survival). It is possible that the spiral scanor molecular analyses of sputum may allow earlier detection, withcancers that can be more readily resected. However, no screening toolhas yet been discovered, due in particular to the risks attached to lungbiopsies and to surgery, especially in patients who are smokers.

Without treatment, small cell lung cancer is the most aggressive of thelung tumors, with a median survival of only 2 to 4 months. Compared withother types of lung cancer, small cell lung cancer has a tendency towarddissemination before diagnosis, but it is more sensitive to chemotherapyand to radiotherapy.

Non small cell lung cancer (NSCLC) covers various histologies. The mostcommon histologies are epidermoid or squamous carcinoma, adenocarcinomaand large cell carcinoma. These histologies are often classifiedtogether because the approaches to diagnosis, grading, establishment ofprognosis, and treatment are similar. Patients presenting a resectablecancer can be cured by surgery or surgery with adjuvant chemotherapy.Local control of the disease can be carried out with radiotherapy in alarge number of patients with a nonresectable cancer. Patientspresenting a locally advanced and nonresectable disease may have along-term survival with radiotherapy combined with chemotherapy.Patients presenting advanced metastatic disease may experience animprovement of symptoms and of survival with chemotherapy. At diagnosis,patients with NSCLC can be divided up into three groups of similartreatment. The first group of patients comprises surgically resectabletumors (generally stage 1, stage II and certain stage III patients).This group has the best prognosis. Patients presenting a resectablecancer with medical contraindications for surgery are candidates forcurative radiotherapy.

The second group includes patients with a locally (T3-T4) and/orregionally (N2-N3) advanced lung cancer.

The third group includes patients with remote metastases (MI) which werefound at the time of diagnosis. This group can be treated withpalliative radiotherapy or chemotherapy.

Multiple studies have tried to identify determining prognostic factors(Albain K S, Crowley J J, LeBlanc M, et al.: Survival determinants inextensive-stage non-small-cell lung cancer: the Southwest Oncology Groupexperience. J Clin Oncol 9 (9): 1618-26, 1991; Macchiarini P, FontaniniG, Hardin M J, et al.: Blood vessel invasion by tumor cells predictsrecurrence in completely resected TI NO MO non-small-cell lung cancer. JThorac Cardiovasc Surg 106 (1): 80-9, 1993; Ichinose Y, Yano T, Asoh H,et al.: Prognostic factors obtained by a pathologic examination incompletely resected non-small-cell lung cancer. An analysis in eachpathologic stage. J Thorac Cardiovasc Surg 110 (3): 601-5, 1995; MartiniN, Bains M S, Burt M E, et al.: Incidence of local recurrence and secondprimary tumors in resected stage I lung cancer. J Thorac Cardiovasc Surg109 (1): 120-9, 1995; Fontanini G, Bigini D, Vignati S, et al.:Microvessel count predicts metastatic disease and survival in non-smallcell lung cancer. J Pathol 177 (1): 57-63, 1995). The factors whichcorrelate with an unfavorable prognosis include the following:

-   -   Presence of pulmonary symptoms.    -   Large size of the tumor (>3 centimeters).    -   Non epidermoid histology.    -   Node metastases in the lymph nodes, established by TNM.    -   Vascular invasion.

Similarly, contradictory results concerning the prognostic importance ofthe abnormal expression of a certain number of proteins in lung cancershave been reported. For patients with an inoperable cancer, theprognosis is compromised by a poor general condition and a weight lossof >10%.

Since the treatment is not satisfactory for almost all patients withNSCLC, it is necessary to discovery new therapeutic targets, and newtools for early diagnosis.

Prostate cancer is the most common cancer in men over the age of 50 andrepresents the second most common cause of death due to cancer in men inthe developed world, after lung cancer.

Its incidence increases with age. In France, the overall incidence in1990 was 71.4 per 100 000 (2.6 in the 35-49 year old age range; 133.8 inthe 50-69 year old age range; 726.9 in the 70 year old age range). Theaverage age for prostate cancer is around 70 years old, but some men areaffected at an earlier age.

The 23% increase in mortality linked to prostate cancer over the pasttwenty years reflects the increase in life expectancy and the morefrequent recognition of prostate cancer as principle cause of death. InFrance, the overall mortality due to cancer was 33.4 for 100 000 in1990, i.e. more than 9000 deaths per year. Prostate cancer represents3.4% of all deaths and 10.7% of deaths due to cancer.

Prostate cancer often develops very slowly, and remains localized at thebeginning. When the cancer progresses, it can spread out of the prostateby direct invasion of the tissues and organs located close to theprostate, and it can spread into other organs remote from the prostate.

Prostate Specific Antigen (PSA) is a tumor marker used for detectingprostate cancer. The level of PSA in the blood is expressed in nanogramsper milliliters (ng/ml) and is considered to be normal if the level isless than 4 ng/ml. The higher the PSA level in the case of prostatecancer, the greater the risk of remote spreading of the cancer, whichusually means a decrease in the chances of recovery or long-termsurvival. Nevertheless, PSA is not the ideal marker: this is becausecertain cancers detected by an increased PSA level could have had a veryslow progression, without the need for treatment. It is thereforeessential to discover new diagnostic tools (for diagnosis at the tissuelevel or at the level of biological fluids), in order to preciselydetect cancers with an aggressive potential, to redistinguish fromcancers with a very slow progression.

In order to specify the diagnosis, endorectal echography of the prostateis used, which can very precisely guide a needle in order to takesamples at a given site in the prostate. Only biopsy can confirm cancer,since the cancer cells are visible under the microscope. Biopsies aretherefore of essential importance for determining the prognosis of thedisease.

The reference treatment is radical prostatectomy. This operation removesthe entire prostate and the seminal vesicles. It is only carried out ifthe cancer does not exceed the limits of the prostate. Approximately 10%of patients will develop a local recurrence within 5 years following aradical prostatectomy for localized prostate cancer.

Radiotherapy is used to treat cancers which are localized in theprostate, or which have reached the neighboring tissues. It can be usedto reduce the tumor volume or to prevent local complications.

The objective of hormone treatment is to oppose the action of the malehormones (androgens) which stimulate the prostate. Thus, decreasing thelevel of testosterone, the main male hormone, blocks the proliferationof the cancer cells and reduces the volume of the prostate. Hormonetreatment only has a transient effect; it blocks the proliferation ofthe cancer without curing it.

Chemotherapy is used in prostate cancer when the latter has progressedwith spreading outside the prostate and no longer responds to hormonetreatment. Chemotherapy reduces the tumor growth and can reduce the painassociated with the cancer.

The treatments mentioned above may have a certain number of sideeffects, including urinary incontinence, impotence, intestinal problems(diarrhea, colitis), and urinary problems occurring essentially duringthe treatment (frequency of urination, weakened urine stream, urgentneed to urinate, burning sensation when urinating, presence of blood inthe urine). Long-term hormone treatment can lead to osteoporosis withbone becoming brittle.

New targeted therapeutics could make it possible to prevent a certainnumber of these side effects, and to improve the effectiveness of thetreatment for prostate cancer. New diagnostic and prognostic tools couldmake it possible to differentiate between slowly progressing cancers andaggressive cancers with metastatic potential.

In clinical practice, the characterization of a tumor in terms ofmalignancy is carried out, after the tumor has been discovered, byhistological methods in specialized laboratories. A set of parameterssuch as the size of the tumor, its histopathological grade, associatedinflammation and lymph node invasion are used to decide on the therapyand to estimate the prognosis of the disease.

Markers which make it possible to distinguish between tumor cells andnormal cells have been sought and studied for years, for many cancers,including breast, thyroid, lung and prostate cancer. They would make itpossible to diagnose the disease early, to establish the prognosis forsaid disease and the sensitivity to treatment, and to monitor theprogression of said disease. Up until now, the candidate markers whichhave been identified and studied have been oncogenes, tissue markers andmarkers associated with angiogenesis or with the metastatic capacitiesof the tumor. Currently, the breast cancer markers identified are mainlyused for therapeutic follow-up. There is no validated biological testfor the early diagnosis or for the screening of breast cancer, nor isthere one for many other cancers (for the mass screening of colorectalcancer, there is the detection of hemoglobin in the stools). PSA can beused to aid the diagnosis and to indicate the need for a prostate biopsyin the case of prostate cancer. Immunoassay for calcitonin in the plasmais an excellent marker for medullary thyroid cancers. In certaincountries, it may be used for screening for prostate cancer, but it hasnot been validated on a population base for this indication. Only thedetection of oestrogen receptors on breast tumor tissue makes itpossible to determine whether the breast tumors will or will not behormone-sensitive. Detection of the HER-2/neu receptor in breast cancersmakes it possible to determine whether the tumor is sensitive toHerceptin.

A limited number of antigenic markers, in particular CA 15-3 (Basuyau,J. P., M. P. Blanc-Vincent, J. M. Bidart, A. Daver, L. Deneux, N. Eche,G. Gory-Delabaere, M. F. Pichon, and J. M. Riedinger. 2000. [Standards,Options and Recommendations (SOR) for tumor markers in breast cancer.SOR Working Group]. Bull Cancer. 87:723-37) has been identified in thecase of cancerous breast cells. It is common practice for this marker tobe used for patient follow-up, in particular for the detection ofrecurrence, but, because of its low sensitivity, it is not proposed ineither a screening test or diagnostic test.

For several years, studies relating to the antigens associated withbreast cancer have been developed, not in order to look for markers, butin order to look for targets for immunotherapy. These studies range fromthe demonstration of a humoral immunity against T/Tn antigens (Springer,G. F. 1997. Immunoreactive T and Tn epitopes in cancer diagnosis,prognosis, and immunotherapy. J Mol. Med. 75:594-602), to the morerecent discovery of antibodies and of T-cell responses directed againstp53 (Gnjatic, S., Z. Cai, M. Viguier, S. Chouaib, J. G. Guillet, and J.Choppin. 1998. Accumulation of the p53 protein allows recognition byhuman CTL of a wild-type p53 epitope presented by breast carcinomas andmelanomas. J. Immunol. 160:328-33) and HER-2/neu (Disis, M. L., and M.A. Cheever. 1997. HER-2/neu protein: a target for antigen-specificimmunotherapy of human cancer. Adv Cancer Res. 71:343-71).

More recently, a series of new potential antigens has been revealed bythe SEREX (serological expression cloning) approach, based on theconstruction of cDNA libraries of tumor cells and screening with theautologous serum. Serological breast cancer library screening has thusmade it possible to reveal the ING1 antigen (Jager, D., E. Stockert, M.J. Scanlan, A. O. Gure, E. Jager, A. Knuth, L. J. Old, and Y. T. Chen.1999. Cancer-testis antigens and ING1 tumor suppressor gene product arebreast cancer antigens: characterization of tissue-specific ING1transcripts and a homologue gene. Cancer Res. 59:6197-204.), and then anew differentiation antigen, NY-BR-1, expressed, according to theauthors, in 80% of breast cancers and inducing the production of IgGantibodies in the patients (Jager, D., E. Stockert, A. O. Gure, M. J.Scanlan, J. Karbach, E. Jager, A. Knuth, L. J. Old, and Y. T. Chen.2001. Identification of a tissue-specific putative transcription factorin breast tissue by serological screening of a breast cancer library.Cancer Res. 61:2055-61). This type of approach, which has mainly beenused to search for targets that can potentially be used for developingvaccines, does not, a priori, exclude the antigens present in normaltissue (this is the case of NY-BR-1), nor those recognized by a limitednumber of sera from patients (2/14 for ING1); they cannot therefore beexploited for a screening or early diagnosis strategy. By means of thesame approach, other antigens inducing a humoral immune response inpatients have been revealed, such as NY-BR-62, NY-BR-85 and the D52protein. These antigens are thought to be overexpressed respectively in60%, 90% and 60% of breast cancers (Scanlan, M. J., and D. Jager. 2001.Challenges to the development of antigen-specific breast cancervaccines. Breast Cancer Res. 3:95-8.).

The molecular phenomena which result in the development of cancers, andmore particularly of a breast cancer, involve modifications of thestructure and of the expression of oncogenes (such as ras) and mutationsof tumor-suppressor genes such as p53. The growth of tumor cells in themajority of breast cancers is dependent on oestrogenic hormones(oestradiol and progesterone) and on growth factors which controlproliferation, migration and apoptosis. The growth of prostate cancersis androgen-dependent. The growth of certain thyroid cancers is thyroidstimulating hormone (TSH)-dependent. These growth factors eitherstimulate or inhibit the proliferation, migration and differentiation ofthe tumor cells in such a way as to act in concert so as to promote thegrowth of the cancer and the metastases. For example, insulin-typegrowth factors, transforming growth factor α (TGF-α) and fibroblastgrowth factors (FGFs) can all stimulate the proliferation of breastcancer cells, while mammary-derived growth factor inhibiter (MDGI) andtransforming growth factor β (TGF-β) inhibit their growth.

In patent application WO2004/040312, the Applicants have described theuse of NGF as a tumor marker and as a therapeutic target. Thus, NGF isproduced by breast cancer cells, whereas corresponding normal mammaryepithelial cells do not produce it. Furthermore, NGF stimulates thesurvival and proliferation of cancerous mammary epithelial cells,whereas it has no effect on normal mammary epithelial cells.

The NGF gene encodes a protein precursor called ProNGF (26 kDa) which,by enzymatic cleavage, generates NGF (13.6 kDa) (Seidah et al., 1996,Biochem J., 314: 951-960). The main source of NGF in mammals is thesubmaxillary gland, which contains only NGF and very little ProNGF. Fora long time, the only role given to ProNGF was a role as a metabolicprecursor with a very transient life time.

More recently, it has been shown, in various tissues, that ProNGF isfound in an amount of greater than that of NGF (Bierl et al., 2005,Neurosci Lett, 380: 133-137). Hasan et al. have described that ProNGFcan be secreted, which means that it can potentially act in anautocrine, paracrine or even endocrine manner (2003, J Neurobiol, 57:38-53). Finally, it has recently been demonstrated that ProNGF has, atthe surface of neuronal cells, a high-affinity binding site throughwhich it has specific effects, different than those of NGF. Thus, ProNGFis capable of binding to sortilin, a glycoprotein of 100 kDa (Mazella etal., 1998, J Biol Chem, 273: 26273-26276), and of inducing neuronal cellapoptosis in vitro (Nykjaer et al., 2004, Nature, 427: 843-848). Patentapplications WO 2004/056385 and WO 2005/076695 show that the interactionbetween a neurotrophin (NGF or ProNGF) can be modified using specificcompounds, such a modulation allowing the treatment of pain associatedwith certain diseases of the urogenital system or of patients whosenervous system has been damaged. An in vivo pro-apoptotic activity hasalso been attributed to ProNGF (Pedraza et al., 2005, Am J Pathol, 166:533-543). It is clear that there is a separation in terms of theexpression and the biological effects of NGF and of ProNGF, which appearto be different.

More generally, propeptides have for a long time been considered to beonly metabolic precursors; a certain number of recent examples, inparticular in the neuropeptide field, indicate that, in certainsituations, propeptides have their own biological activity, dissociatedfrom that of the mature peptide that they are able to generate. This isclearly the case of ProNGF, which can be secreted by the cell, has itsown receptors and is shown to have biological effects different thanthose of NGF on neurons. ProNGF thus constitutes a specific molecularand biological entity different than NGF. Admittedly, the sequence ofNGF is contained in that of ProNGF, but their isoelectric points andmolecular weights are different, as are their biological activities.

The Applicants have now demonstrated, surprisingly, that epithelialcancer cells, and in particular epithelial breast, thyroid, lung andprostate cancer cells, themselves produce and secrete ProNGF, in notableamounts, whereas normal epithelial cells of the same organs do notproduce it, such that ProNGF can be used as a tumor marker or else as atherapeutic target. The Applicants have also established that ProNGFoverexpressed in cancer cells has a prometastatic activity for thesecells, whereas NGF, for its part, has an anti-apoptotic and mitogenicactivity.

Thus, a first subject of the present invention is a method for in vitrodiagnosis of cancer, and in particular breast, thyroid, lung or prostatecancer, by determining the presence of ProNGF in biological samplesderived from patients suspected of suffering from cancer, and inparticular breast, thyroid, lung or prostate cancer.

The method of the invention thus makes it possible to diagnose thecancer, and in particular breast, thyroid, lung or prostate cancer, bymeans of a simple test consisting in searching for the presence ofProNGF in a biological sample taken from a patient, or in the patient'stumor in vivo. The Applicants have shown, unexpectedly, that cancerouscells produce ProNGF, whereas the corresponding noncancerous cells areincapable of doing so, as will be demonstrated in more detailhereinafter. Thus, the determination of the presence of ProNGF in thesample makes it possible to conclude that the pathology being sought ispresent, the absence of ProNGF making it possible to conclude that thepathology is absent. The presence of ProNGF in the tumors may also beshown in vivo, in situ in the tumors.

In order to show the presence of ProNGF in a tumor in vivo, any imagingmethod known to those skilled in the art can be used. For this, aspecific ProNGF binding partner can be coupled to an imaging tracer. Thespecific ProNGF binding partners are any partner capable of binding toProNGF. By way of example, mention may be made of antibodies, antibodyfractions, receptors and any other molecule capable of binding toProNGF.

The binding-partner antibodies are, for example, either polyclonalantibodies or monoclonal antibodies.

The expression “coupling of binding partners to an imaging tracer” isintended to mean the attachment of a tracer that can be detected by anyimaging method known to those skilled in the art, and that can directlyor indirectly generate a detectable signal. Thus, the tracer may be aradioactive tracer such as technetium-99. In this case, the organaffected by the primary cancer or the metastases will bind the ProNGFand its tracer. The radiation emitted by the organ can be filmed by aspecial camera, for example a gamma-camera. The apparatus collects thescintillations generated by the radioactive substance and thus makes itpossible to visualize the organ.

In another method of the invention, the tracer comprises apositron-emitting radioactive substance (fluorine 18). The images willthen be captured by a Positron Emission Tomography system.

In another preferred method of the invention, the ProNGF partner may becoupled to nanoparticles. By way of example, they may be supramagneticnanoparticles; for example, anionic magnetic nanoparticles for use indirect cell labeling and in vivo detection by nuclear magnetic resonanceimaging. They may also be gold nanoparticles. By virtue of the methodsof the invention that make it possible to detect ProNGF in vivo, theareas of the body where there has been binding of the ProNGF bindingpartner, cancers producing ProNGF, in particular breast, prostate,thyroid and lung cancers, and also the locations of their remotemetastases and the lymph node involvements may be visualized.

The determination of the presence of ProNGF in vitro can be carried outby direct detection of the ProNGF, by culturing cells sensitive toProNGF, or by any other method for determining the presence of a proteinin the sample, known to those skilled in the art.

The determination of the presence of ProNGF by direct detection of theProNGF constitutes a specific embodiment of the invention.

The term “direct detection of the ProNGF” is intended to mean thedemonstration of the ProNGF itself in the biological sample.

The direct detection of the ProNGF in the biological sample can becarried out by any means known to those skilled in the art, such as, forexample, by immunoassay or by mass spectrometry, which constitutes aspecific embodiment of the invention.

The immunoassay may be any assay widely known to those skilled in theart involving immunoreactions, namely reactions between the ProNGF and aspecific ProNGF binding partner.

The specific ProNGF binding partners are any partner capable of bindingto ProNGF. By way of example, mention may be made of antibodies,antibody fractions, receptors and any other molecular capable of bindingto ProNGF.

The binding-partner antibodies are, for example, either polyclonalantibodies or monoclonal antibodies.

The polyclonal antibodies may be obtained by immunization of an animalwith ProNGF, followed by recovery of the desired antibodies in purifiedform, by taking a sample of the serum of said animal and separating saidantibodies from the other serum constituents, in particular by affinitychromatography on a column to which an antigen specifically recognizedby the antibodies, in particular ProNGF, is attached. The monoclonalantibodies may be obtained by the hybridoma technique, the generalprinciple of which is summarized below.

Initially, an animal, generally a mouse (or cells in culture in the caseof in vitro immunizations), is immunized with ProNGF, and the Blymphocytes of said animal are then capable of producing antibodiesagainst said antigen. These antibody-producing lymphocytes aresubsequently fused with “immortal” myeloma cells (murine in the example)so as to produce hybridomas. From the heterogeneous mixture of cellsthus obtained, a selection of the cells capable of producing aparticular antibody and of multiplying indefinitely is then made. Eachhybridoma is multiplied in the form of a clone, each one resulting inthe production of a monoclonal antibody whose properties of recognitionwith respect to ProNGF may be tested, for example, by ELISA, by one- ortwo-dimensional immunoblotting, by immunofluorescence, or using abiosensor. The monoclonal antibodies thus selected are subsequentlypurified, in particular according to the affinity chromatographytechnique described above.

The monoclonal antibodies may also be recombinant antibodies obtained bygenetic engineering, by techniques well known to those skilled in theart.

Examples of anti-ProNGF antibodies are known and are available inparticular in the Chemicon catalog.

The specific ProNGF binding partners may be labeled for visualization ofthe ProNGF/binding partner binding when the binding partner is used as adetection reagent, and therefore for the direct detection of ProNGF inthe biological sample.

The term “labeling of the binding partners” is intended to mean theattachment of a label capable of directly or indirectly generating adetectable signal. A nonlimiting list of these labels consists of:

-   -   enzymes which produce a signal detectable, for example, by        colorimetry, fluorescence or luminescence, such as horseradish        peroxidase, alkaline phosphatase, α-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent, luminescent or dye compounds,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I, and    -   fluorescent molecules such as Alexas or phycocyanins.

Indirect systems may also be used, for instance ligands capable ofreacting with an antiligand. The ligand/antiligand pairs are well knownto those skilled in the art, which is the case, for example, of thefollowing pairs: biotin/streptavidin, hapten/antibody, antigen/antibody,peptide/antibody, sugar/lectin, polynucleotide/sequence complementary tothe polynucleotide. In this case, it is the ligand which carries thebinding partner. The antiligand may be directly detectable by virtue ofthe markers described in the previous paragraph or may itself bedetectable by means of a ligand/antiligand.

These indirect detection systems may, under certain conditions, resultin an amplification of the signal. This signal amplification techniqueis well known to those skilled in the art, and reference may be made toprior patent applications FR98/10084 or WO-A-95/08000 by one of theApplicants, or to the article J. Histochem. Cytochem. 45: 481-491, 1997.

Depending on the type of labeling used, those skilled in the art willadd reagents for visualizing the labeling.

By way of example of immunoassays as defined above, mention may be madeof “sandwich” methods such as ELISA, IRMA and RIA, “competition” methodsand direct immunodetection methods such as immunohistochemistry,immunocytochemistry, Western blotting and Dot blots.

In the case of the “competition” methods, the ProNGF is labeled asdescribed above for the binding partner.

Mass spectrometry may also be used for direct detection of ProNGF in thebiological sample. The principle of spectrometry is widely known tothose skilled in the art and is described, for example, in Patterson,S., 2000, Mass spectrometry and proteomics. Physiological Genomics 2,59-65.

To do this, the biological sample, which may or may not be pretreated,is passed through a mass spectrometer and the spectrum obtained iscompared with that of ProNGF. An example of a pretreatment of the sampleconsists in passing it over an immunocapture support comprising one ofthe ProNGF binding partners, for example an antibody directed againstProNGF. Another example of pretreatment of the sample may beprefractionation of the biological sample, in order to separate theproteins in the sample from one another. In techniques well known tothose skilled in the art, the predominant proteins in the sample may,for example, first of all be depleted.

The biological sample used for the direct detection of ProNGF, which maycontain ProNGF as such, may consist of biological fluid or a tissueoriginating from the biopsy of the tumor or of the metastases of thepatient under consideration.

By way of biological fluid, mention may be made of whole blood andderivatives thereof, such as serum or plasma, bone marrow, milk,cerebrospinal fluid, urine and effusions. Blood or derivatives thereofis/are preferred.

For the detection of ProNGF, the biological fluid, which constitutes aspecific embodiment of the invention, may require a specific treatment.This is because the biological fluid may contain ProNGF as such, or elseit may contain circulating tumor cells which contain ProNGF, and/orcirculating tumor cells which are capable of secreting ProNGF.

Thus, according to one embodiment of the invention, the biological fluidis pretreated in order to isolate the circulating tumor cells containedin said fluid.

The expression “to isolate the circulating tumor cells” is intended tomean to obtain a cell fraction enriched in circulating tumor cells.

The treatment of the fluid in order to isolate the circulating tumorcells may be carried out by cell sorting in a flow cytometer, byenrichment on Ficoll, by enrichment using magnetic beads coated withspecific antibodies, or by any other method of specific enrichment knownto those skilled in the art.

In the case of blood or bone marrow as biological fluid, the circulatingtumor cells may be isolated by virtue of a technique of cell separationon Ficoll associated with depletion of the blood cells using anti-CD45antibodies coupled to magnetic beads (Dynal Biotech ASA, Norway).

The direct detection of ProNGF can then be carried out directly oncirculating tumor cells isolated from the biological fluid, for exampleby immunocytochemical labeling of these cells with an anti-ProNGFantibody, after having deposited the circulating tumor cells onto aslide by cytospin. The direct detection of ProNGF can also be carriedout directly in the circulating tumor cells by using the flow cytometrymethod as described in Métézeau P, Ronot X, Le Noan-Merdrignac G,Ratinaud M H, La cytométrie en flux pour I'étude de la cellule normaleou pathologique [Flow ctyometry for studying normal or pathologicalcells] (Tome I), published by Medsi-MacGrawhill.

Under these conditions, said circulating cells can be treated underconditions which allow the ProNGF to be blocked inside said cells. Sucha treatment is described, for example, in Intracellular Flow Cytometry,Applied reagents and Techniques, pp 1-21, BD Pharmingen.

The detection of ProNGF is then carried out after having made themembrane of the cells permeable so as to enable the specific ProNGFbinding partners to enter.

The direct detection of ProNGF using the circulating cells can also becarried out by means of the method described in patent application WO03/076942 filed by one of the Applicants.

The direct detection of ProNGF in the tumor cells can also be carriedout in the culture medium of said cells, after having cultured themunder conditions such that they secrete ProNGF.

These culture conditions are conventional conditions, such as 37° C. ina humid atmosphere and at 5% CO₂.

When the biological sample to be tested is tissue originating from thebiopsy of the tumor or of the metastases of the patient, whichconstitutes a specific embodiment of the invention, the direct detectionof ProNGF is carried out directly on the sections obtained, withoutpretreatment of said tissue.

Another method of detecting the presence of ProNGF consists in culturingProNGF-sensitive cells in the presence of the biological sample, whichconstitutes a specific embodiment of the invention.

In this case, the detection of the presence of ProNGF in a biologicalsample is demonstrated by the reaction of the ProNGF-sensitive cells.

The term “ProNGF-sensitive cells” is intended to mean any cellstimulated in the presence of ProNGF (growth, apotosis, etc.).

By way of ProNGF-sensitive cells, mention may be made of cells ofneuronal origin, such as, for example, PCl2 cells (Pedraza et al. Am. J.Pathol. 2005, 166, 533-543).

The biological sample that can be used for detecting the presence ofProNGF by culturing ProNGF-sensitive cells may be any sample asdescribed above.

Thus, the biological sample may consist of biological fluid, whereappropriate pretreated in order to isolate the circulating tumor cells,which themselves may subsequently be cultured under conditions such thatthey secrete ProNGF, as described above.

The method of the invention may be used both for early diagnosis and forscreening, therapeutic follow-up, prognosis and diagnosing relapses incancer, and in particular breast, thyroid, lung or prostate cancer,since only the cancerous cells produce ProNGF and this productiondepends on the grade of the cancer.

Thus, another subject of the invention consists of the use of the methodof the invention in early diagnosis, screening, therapeutic follow-up,prognosis and diagnosing of relapses in cancer, and in particularbreast, thyroid, lung or prostate cancer.

In addition to a tumor marker role, ProNGF may also have a role as atherapeutic target. In fact, due to the ability of cancerous cells, inparticular breast, thyroid, lung or prostate cancer cells, to produceProNGF, whereas normal cells do not produce it, the remote disseminationof breast, thyroid, lung or prostate cancer cells and the invasion ofthe cells can be blocked by a ProNGF inhibitor capable of blocking theactivity of ProNGF.

Moreover, ProNGF, alone or in combination with other molecules, may beused as a therapeutic target for targeting therapeutic tools. Thetherapeutic tools may, in this case, be activatable nanoparticles,cytotoxic agents, or any other molecule which makes it possible todestroy cancerous cells.

The ProNGF inhibitors may therefore be used as drugs.

Thus, a subject of the present invention is also the use of a ProNGFinhibitor for preparing a drug, in particular in the treatment of cancerand more particularly breast, thyroid, lung or prostate cancer.

According to a specific embodiment of the invention, said drug can beused for blocking cell migration or the invasion of tumor cells inpatients suffering from cancer, and more particularly breast, thyroid,lung or prostate cancer.

The term “cell migration” is intended to mean remote dissemination inpatients suffering from cancer, and more particularly breast, thyroid,lung or prostate cancer (metastases), and the term “invasion” isintended to mean the local penetration of the cancerous cells.

The pharmaceutical compositions comprising, as active ingredient, atleast one ProNGF inhibitor, optionally in combination with apharmaceutically acceptable excipient, are also included in theinvention.

The pharmaceutical compositions that can be used against cancercomprise, as active ingredient, at least one ProNGF inhibitor capable ofblocking cell migration or invasion.

The term “ProNGF inhibitor” is intended to mean direct inhibitors ofProNGF, i.e. inhibitors that block the biological activity of theprotein, such as the ProNGF binding partners, and also inhibitors ofProNGF receptors or any inhibitor of ProNGF signaling pathways, and alsoany molecule that can specifically bind to ProNGF, irrespective ofwhether or not there is blocking of the biological activity.

An example of a ProNGF receptor expressed in both tumoral and normalbreast epithelial cells comprises Sortilin.

The specific ProNGF binding partners suitable as active ingredient arein particular as defined above in the immunoassays and may be any otherpartner known to those skilled in the art capable of blocking cellmigration or invasion. According to a specific embodiment, the specificpartner capable of blocking the migration or invasion of breast cancercells is an anti-ProNGF antibody.

Another example of a direct inhibitor of ProNGF comprises the analogs,in soluble form, of ProNGF receptors, such as Sortilin analogs insoluble form, which also constitutes an embodiment of the invention.

The expression “inhibitor of ProNGF receptors expressed by both tumoraland normal breast epithelial cells” is intended to mean any moleculeswhich block the activity of ProNGF, for example either via said receptoror by blocking its production. The blocking of ProNGF activity via saidreceptor may be carried out by preventing the binding of ProNGF to saidreceptor, for instance using an agent capable of binding to saidreceptor, which thus takes the place of the ProNGF. An example of suchan inhibitor comprises a ProNGF-derived peptide which has conserved theproperties of binding to said receptor or an antibody directed againstthe receptor. The activity of ProNGF may also be blocked by blocking theproduction of said receptor using inhibitors of the mRNA of saidreceptor or of the gene encoding said receptor. As an inhibitor of thereceptor mRNA, use may be made of a synthetic fragment of this mRNA. Infact, interference RNA technology (Small Interference RNA or siRNA) isbased on the use of a double-stranded RNA oligonucleotide correspondingto a short sequence of the cellular mRNA to be inhibited. Thisoligonucleotide in the form of a duplex (possibly introduced into aplasmid), after it has entered the cell, is dealt with by the Dicer/Riscenzyme system, which will bring about the degradation of thecorresponding cellular mRNA (Dykxhoom D et al., 2003, Nature Reviews,vol. 4, p457-467). By way of inhibitor of the gene encoding thereceptor, mention may be made of an antisense oligonucleotide of saidreceptor. This oligonucleotide can be readily prepared by those skilledin the art (Lefrancois S et al. Biol. Proced. online. 2005, 7(1):17-25).

According to a specific embodiment of the invention, the ProNGFinhibitor is an siRNA of the ProNGF receptor.

The expression “ProNGF signaling pathway inhibitor” is intended to meanany molecule which blocks the biological activity of ProNGF, such as itsactivity on cell migration and invasion.

In order to target the therapeutic action of the various inhibitors, thelatter may be placed under conditions such that they specificallypenetrate into the cells of interest, for instance tumor cells, whichconstitutes another embodiment of the invention.

To this effect, they may, for example, be attached to a partner whichallows such penetration, for instance a carrier molecule, a polymer suchas the polymers used in gene therapy or else a viral vector such asadenoviruses and poxviruses, also used in gene therapy.

For example, in the case of breast cancer, the carrier molecule may bean anti-MUC1 antibody or an anti-epithelial cell antibody, oralternatively an anti-HER/2/neu antibody. In the case of thyroid cancer,the carrier molecule may be an anti-thyroglobulin antibody or ananti-epithelial cell antibody. In prostate cancer, the carrier moleculemay be an anti-PSA antibody or an anti-epithelial cell antibody. In thecase of lung cancer, the carrier molecule may be an anti-epithelial cellantibody.

The ProNGF signaling pathway inhibitors may also be attached toanticancer agents. The term “anticancer agent” is intended to mean acompound which will be toxic for the cancerous cell. For example, theProNGF partners may be associated with therapeutic nanoparticles, whichmay allow the targeted destruction of the tumor when they are activated.In another method of the invention, the ProNGF partner may be coupled toa cytotoxic agent, or to a molecule for blocking a carcinogenic process.

Preferably, when the pharmaceutical composition comprises, as activeingredient, a ProNGF inhibitor such as a direct inhibitor or a ProNGFsignaling pathway inhibitor, the latter are placed under conditions suchthat they specifically penetrate into the tumor cells of interest, theProNGF mRNA inhibitors and the inhibitors of the gene encoding NGFhaving the ability to naturally penetrate into said cells.

The amount and the nature of the excipient may be readily determined bythose skilled in the art. They are chosen according to thepharmaceutical form and the method of administration desired.

The pharmaceutical compositions of the invention are suitable for oral,sublingual, subcutaneous, intramuscular, intratumoral, intravenous,topical, local, intratracheal, intranasal, transdermal, rectal,intraocular or intra-auricular administration, it being possible forsaid active ingredient to be administered in unit administration form.

The unit administration forms may, for example, be tablets, gelcapsules, granules, powders, injectable oral solutions or suspensions,patches, sublingual, buccal, intratracheal, intraocular, intranasal orintra-auricular administration forms, forms of administration byinhalation, topical, transdermal, subcutaneous, intramuscular,intratumoral or intravenous forms, rectal administration forms, orimplants. For topical administration, creams, gels, ointments, lotionsor eye lotions can be envisioned.

These galenical forms are prepared according to the usual methods in thefields under consideration.

Said units forms contain a dosage so as to allow daily administration offrom 0.001 to 10 mg of active ingredient per kg of body weight,according to the galenical form.

There may be specific cases where higher or lower dosages areappropriate; such dosages do not depart from the scope of the invention.According to the usual practice, the dosage appropriate for each patientis determined by the physician according to the method of administrationand the weight and response of the patient.

According to another embodiment, the present invention also relates to amethod for treating breast cancer, which comprises the administration,to a patient, of an effective dose of a ProNGF binding partner or of aProNGF inhibitor.

The invention will be understood more clearly from the followingexamples, given by way of nonlimiting illustration, and also from tables1 and 2 and from attached FIGS. 1 to 10, on which:

FIG. 1 represents a photograph of a Western blot showing the productionof ProNGF by cancerous mammary epithelial cells (MCF-7, T47-D, BT-20 andMDA-MB-231), but not by normal cells (NMEC cells), actin having beenused as positive control,

FIG. 2 represents a photograph of a Western blot showing the presence ofProNGF in the cancerous breast biopsies (ST-1 to -4) but not in thenormal biopsies (SS-1 to -4), actin having been used as normalizationcontrol,

FIG. 3 represents a photograph of a Western blot showing:

FIG. 3A: the secretion of ProNGF by MCF-7 cancerous cells (concentratedconditioned medium lane), the two lanes on the right being controllanes,

FIG. 3B: that the concentrating/desalifying unit used for the experimentof FIG. 3A was capable of retaining NGF,

FIG. 4 represents a photograph of a Western blot showing the presence ofProNGF in the sera of mice injected with cancerous cells and its absencein the sera of normal mice,

FIG. 5 represents graphs of detection by mass spectrometry of ProNGF inthe medium conditioned with the MCF-7 cells (FIGS. 5A, 5C and 5E) or ofrecombinant ProNGF (FIGS. 5B and 5D), giving the relative abundance as afunction of the mass,

FIG. 6 represents a photograph of a Western blot showing the presence ofSortilin in the cancerous mammary epithelial cells (MCF-7, T47-D, BT-20and MDA-MB-231) and the normal cells (NMEC cells), actin having beenused as positive control,

FIG. 7 represents a graph giving the number of MDA cells which aregrowing in EMEM culture medium after 24, 48 and 96 hours of culture,having been transfected either with culture medium alone (Mock), or witha negative control interfering RNA (siGFP, double-stranded RNA moleculecomposed of the partially complementary sequences SEQ ID No.1 and SEQ IDNo. 2 in which the nucleotide N represents thymine T), or with aninterfering RNA which decreases the expression of Sortilin (siSORT,double-stranded RNA molecule composed of the partially complementarysequences SEQ ID No.3 and SEQ ID No. 4 in which the base N representsthymine T),

Sequence siGFP: SEQ ID No. 1 5′-GCUGACCCUGAAGUUCAUCNN-3′ SEQ ID No. 25′-GAUGAACUUCAGGGUCAGCNN-3′ Sequence siSORT: SEQ ID No. 35′-GGUGGUGUUAACAGCAGAGNN-3′ SEQ ID No. 4 5′-CUCUGCUGUUAACACCACCNN-3′

FIG. 8 represents a graph giving the percentage cell migration usingMCF-7 cells bathed either in starving medium alone, or in starvingmedium supplemented with 200 ng/ml of ProNGF, or in starving mediumsupplemented with 200 ng/ml of ProNGF and 20 μM of galardin, or instarving medium supplemented with 20 μM of galardin, or in starvingmedium supplemented with 200 ng/ml of NGF,

FIG. 9 represents a graph giving the invasion index using MCF-7 cellsbathed either in starving medium alone, or in starving mediumsupplemented with 200 ng/ml of ProNGF, or in starving mediumsupplemented with 200 ng/ml of ProNGF and 20 μM of galardin, or instarving medium supplemented with 20 μM of galardin, or in starvingmedium supplemented with 200 ng/ml of NGF, and

FIG. 10 plotted from the Western blot photographs shows the decrease inexpression of ProNGF in the MCF-7 cells following transfection of aProNOF interfering RNA (siProNGF), after 24 or 48 hours of culture.Actin was used as positive control for validating the equal loading. TheMCF-7 cells were transfected with interfering RNAs (siRNAs) directedagainst GFP (siGFP, composed of the partially complementary sequencesSEQ ID No.1 and SEQ ID No. 2), or against ProNGF (siProNGF,double-stranded RNA molecule composed of the partially complementarysequences SEQ ID No.5 and SEQ ID No. 6 in which the base N representsthymine T). The relative amount of ProNGF detected by blotting wasevaluated with the Quantityone software (Bio-Rad), related to the equalloading and presented in the form of a histogram where 100% is assignedto the siGFP control condition.

Sequence siGFP: SEQ ID No. 1 5′-GCUGACCCUGAAGUUCAUCNN-3′ SEQ ID No. 25′-GAUGAACUUCAGGGUCAGCNN-3′ Sequence siProNGF: SEQ ID No. 55′-CAGUGUAUUCAAACAGUAUNN-3′ SEQ ID No. 6 5′-GUACUGUUUGAAUACACUGNN-3′

EXAMPLE 1 Cell Culture

1.1. Material

The cells used are established breast cancer cell lines (BT-20, MCF-7,MDA-MB-231, T-47D) obtained from the ATCC (American Type CultureCollection) and NMEC (Normal Mammary Epithelial Cells) in primaryculture, obtained from patients having undergone a mammoplasty at theCentre Oscar Lambret in Lille. The MCF-7, MDA-MB-231 and T-47D areepithelial cells which originate from pleural effusions in patientssuffering from adenocarcinoma; the BT-20, for their part, originate froma primary carcinoma. MCF-7 and T-47D are termed “hormone-sensitive”since they express oestrogen receptors, whereas BT-20 and MDA-MB-231 aretermed “hormone-insensitive” since they do not express them.

The biopsies originate from patients having undergone a mammoplasty ofnonmalignant tissue (Professeur Pellerin CHRU, Lille) or canceroustissue exeresis (Docteur Laurent, Croisé Laroche, Marcq en Baroeul).

The immunohistochemistry slides (tissue array) containing spots oftumoral and normal breast tissues originate from Biochain Inc. (Cat. No.T8235731). Other immunohistochemistry slides containing tissues derivedfrom various organs (cancerous tissues and corresponding normal tissues)originate from Superbio Chips (Cat. No. MA (cancer tissue), MAN(corresponding normal tissue) and AA (various normal organs)). Femalesix-week-old SCID (Severe Combined ImmunoDeficiency) mice were obtainedfrom Iffa Credo (France) and acclimatized for at least two weeks. Thesemice were reared at 20-22° C., while maintaining alternating 12 hday/night (light from 6 am to 6 pm), and fed ad libitum, following theguidelines set by Institutional Animal Care.

The ProNGF used is human recombinant NGF sold by SCIL proteins(Germany). The recombinant EGF, HGF and NGF were obtained from R&Dsystems (France). The cortisol, the insulin, the DMSO (dimethylsulfoxide), the cholera toxin, the transferrin, the C2 ceramide analog(N-acetyl-D-sphingosine), and the Hoechst 33258 (bisbenzimide) wereobtained from Sigma (France). EMEM (Eagle's Minimum Essential Medium),DMEM/F12 (Dulbecco's Modified Eagle's Medium), trypsin-EDTA(ethylenediaminetetraacetate), HEPES(N-2-hydroxyethylpiperazine-N′-2′-ethanesulfonic acid), L-glutamine,nonessential amino acids, penicillin/streptomycin and gentamycin aresold by Cambrex (France). The FCS (Foetal Calf Serum) is sold by GibcoInvitrogen Corporation (France). The various plastics used: 1 mLcryotubes, 15 mL and 50 mL centrifuge tubes, 100 mm diameter Petridishes, 75 cm² dishes, 175 cm² dishes and 6-well plates (wells of 35 mmin diameter) are sold by Starstedt (Germany). The Transwell® areobtained from Costar (France). The fibronectin is obtained from BectonDickinson (France). The glycergel is sold by Dako (France). The galardinis obtained from Tebu-Bio (France). The collagen type I is sold byUpstate (United States of America).

1.2. Thawing and Maintenance of Cells

The cell cryotubes are removed from the liquid nitrogen and thawed for 2minutes at 37° C. The suspension is then recovered and transferred intoa 50 mL centrifuge tube containing 19 mL of MEM (Minimum EssentialMedium). The whole is centrifuged at 200 g (15 minutes) in order toremove the traces of DMSO (dimethyl sulfoxide). The cell pellet isrecovered and then taken up with 10 mL of complete medium (compositionbelow) and transferred into a 75 cm² dish. The medium is changed after24 hours, and 24 hours later, the cells are passaged. The cells are thenmaintained for two weeks before any experiments.

The cancer lines are maintained in 75 cm² dishes, on which they adhereand grow as a monolayer in a complete medium: EMEM supplemented with 10%FCS, 20 mM HEPES, 2 g/L of sodium bicarbonate, 2 mM L-glutamin, 1%nonessential amino acids, 40 μg/mL of penicillin/streptomycin and 50μg/mL of gentamycin. The NMEC are maintained in 75 cm² dishes on whichthey adhere and grow as a monolayer in complete medium: DMEM/F12supplemented with 5% FCS, 10 μg/mL of insulin, 5 μM cortisol, 2 ng/mL ofEGF, 0.01 ng/mL of cholera toxin, 100 μg/mL of penicillin and 45 μg/mLof gentamycin.

The cells are cultured in a humid atmosphere at 37° C. and at 5% CO₂.The maintaining medium is changed every two days and the cells arepassaged at confluence.

EXAMPLE 2 Detection of ProNGF

2.1. Methodology

Lysis of Mammary Cells and Biopsies: Protein Extraction

The cells are seeded in three Petri dishes 100 mm in diameter, and then,at 95% confluence, the cells are rinsed twice, on ice, with PBS(Phosphate Buffered Saline): 107 mM KCl, 59 mM KH₂PO₄, 137 mM NaCl, 56mM Na₂HPO₄, and lysed with 100 μL/dish of a lysis buffer containing: 50mM of tris-HCl, pH 7.5, 150 mM of NaCl, 1% Nonidet P-40, 1% SDS (SodiumDodecyl Sulfate), and proteasic activity inhibitors: 1 mM ofphenylmethylsulfonide fluoride, 1 mM of orthovanadate, 1 mM of Na₄P₂O₇,10 μg/mL of leupeptin and 10 μg/mL of aprotinin. The dishes are frozenfor 12 hours and then scraped and the lysates are pooled, heated for 5minutes at 100° C. and centrifuged at 10 000 g (10 min at 4° C.), andthe supernatant is recovered.

The biopsies are weighed and placed, with ten times their volume oflysis buffer (described above), on a wheel (1 hour at 4° C.). They arethen ground, on ice, with a dounce homogenizer, frozen (20 minutes at−80° C.), and thawed on ice. Finally, they are centrifuged at 13 000 g(10 minutes at 4° C.), a lipid disc is removed and the supernatent isrecovered.

The supernatents are assayed by the bicinchoninic acid method comparedwith a range of bovine serum albumin, and then aliquoted in 50 μLaliquots before freezing.

Obtaining Conditioned Medium and Serum

The MCF-7 cells are seeded, in complete medium, into 175 cm² dishes. At90% confluence, these dishes are rinsed twice in starving medium andthen the cells undergo starving for 24 hours with 14 mL of starvingmedium. After this period, the medium conditioned by the cells isrecovered and centrifuged at 1000 g (15 minutes at 4° C.). Thesupernatent is then used directly, or frozen at −20° C.Concentrating/desalifying units with a cut-off threshold of 10 kDa(Centricon Plus 20, Millipore, France) are each loaded with 14 mL ofMCF-7-conditioned medium and then centrifuged at 4 000 g (15 minutes at4° C.). These same units were reloaded a further three times with 14 mLof conditioned medium (which results in a 4-fold concentration of thestarting medium). The units are then desalified with mQ quality water(18.2 osm) by loading 14 mL and centrifuging at 4 000 g (15 minutes at4° C.), this being done twice according to the manufacturer'srecommendations. Finally, the concentrate is recovered by inversion ofthe unit and a centrifugation at 1 000 g (4 minutes at 4° C.); 250 μLare recovered. The concentrated conditioned medium is then aliquoted in50 μL aliquots and frozen at −20° C. A concentration factor of 4×14000/250, i.e. 224 times, is then achieved.

The MDA-MB-231 cells (3×10⁶) are resuspended in PBS and then injectedsubcutaneously in the flanks of eight-week-old SCID mice. The tumorvolume is measured every two days, after two weeks. After seven weeks,the mice are anesthetized with ether, and the blood is collected byintracardiac sampling, before sacrifice of the animals. The blood isleft overnight at 4° C. in order to allow clotting, the serum beingrecovered after centrifugation, assayed and frozen at −20° C.

SDS-PAGE

The aliquots (50 μL) are thawed before use, and taken up with 12.5 μL of5× Laëmmli buffer: 5% SDS, 5% β-mercaptoethanol, 50% glycerol, 50 mMTris, pH 6.8, 0.3% bromophenol blue. The proteins are loaded into thewells of a 12.5% polyacrilamide gel. After migration (at 30 mA, for 5hours) and transfer onto a nitrocellulose membrane (at 200 mA, for 1hour), the quality of the transfer is assessed by staining with Ponceaured.

Immunodetection

The membranes are saturated with 4% bovine serum albumin in TBS (TrisBuffered Saline) solution containing 0.1% Tween 20 (17.54 g NaCl, 2.42 gTris, 2 mL tween 20, QSP 2 L, pH adjusted to 7.4) at ambient temperaturefor 2 hours. After saturation, the membranes are incubated overnight at4° C. with the various primary antibodies: anti-ProNGF (AB9040 fromChemicon) at 1:2000, anti-NGF (SC-548 from Sigma) at 1:2000 oranti-actin (A-2066 from Sigma) at 1:5000 in the saturation solution.After rinsing with TBS 0.1% Tween 20, the membranes are incubated for 1hour at ambient temperature with an anti-rabbit secondary antibody(A-1949 from Sigma, France) coupled to horseradish peroxidase, at 1:20000 in the saturation solution. After rinsing, visualization is carriedout with the ECL system (Pierce Interchim, France) according to therecommended data for use.

Immunohistochemistry

In a first step, the tissue array slides are deparaffinized. For this,they are incubated successively in the following baths for 10 minutes:methycyclohexane (twice), 100% ethanol, 95% ethanol, 70% ethanol, andwater. The slides are then rinsed with TBS 0.1% Tween 20 (TBS-T) for 10minutes, with agitation. The antigens are reactivated in 10 mM citratebuffer, pH6, by heating to 90° C. for 40 min, and then leaving to coolto ambient temperature for 30 min. The endogenous peroxidases areinhibited by incubation in TBS-T containing 3% of H₂O₂, for 5 min. Theslides are then saturated with 3% BSA in TBS-T, for 1 h at 37° C., in ahumid chamber. The slides are then incubated for 2 h with theanti-ProNGF primary antibody (AB9040 from Chemicon) diluted to 1/200 inTBS-T containing 3% BSA (incubation at 37° C. in a humid chamber). Afterthree washes for 10 min with TBS-T, the slides are incubated for 2 h at37° C. in a humid chamber with the anti-rabbit secondary antibodycoupled to horseradish peroxidase (Cat. No. 711-035-152 JacksonImmunoresearch) diluted to 1/400 in the saturation solution. The slidesare washed for three times 10 minutes in TBS-T, and then a further threetimes 10 min in PBS. Visualization is carried out with the Sigma Fastsubstrate (Cat. No. D-4168, Sigma-Aldrich) for 5 min. The staining isstopped by washing in PBS. Counterstaining is performed with Harrishematoxylin (Cat. No. MHS16, Sigma-Aldrich) for 30 sec. After washingwith water and with PBS, the slides are mounted for observation under amicroscope.

Mass Spectrometry (MS)

The MCF-7-conditioned medium is injected onto a C4 LC (LiquidChromatography) nanocolumn (Dionex, France), which makes it possible toseparate the whole proteins according to a gradient of increasinghydrophobicity. Thus, the most hydrophobic proteins are eluted last.Once separated, the whole proteins are ionized by nanoESI (ElectroSprayIonization), and they are then analyzed in an ion trap (LCQ Deca XP⁺™station from Thermo Electron) according to their mass (m) and theircharge (z). We used the SIM (Selected Ion Monitoring) scan technique,which makes it possible to scan only certain ions of interest. Thesemulticharged ions, characteristic of recombinant ProNGF, were determinedbeforehand by nanoLC-nanoESI/MS. Once detected in the conditioned mediumusing SIM of the ProNGF, the corresponding multicharged mass spectrum isresolved so as to find the mass of the protein which generated thisspectrum.

2.2. Results

Detection Using Epithelial Cells

50 μg of total protein extracts of mammary epithelial cells are loadedonto a gel for SDS-PAGE. After transfer onto a nitrocellulose membrane,immunodetection with the AB9040 antibody and the A-2066 antibody (equalloading control) is carried out. NMEC: normal mammary epithelial cells,BT-20, MCF-7, MDA-MB-231 and T-47D are cancerous mammary epithelial celllines.

The results are given in FIG. 1, which represents a photograph of aWestern blot showing the production of ProNGF by the cancerous mammaryepithelial cells (MCF-7, T47-D, BT-20 and MDA-MB-231), but not by normalcells (NMEC cells), actin having been used as positive control.

Detection Using Mammary Biopsies, by Western Blotting

50 μg of protein extracts of biopsies are loaded onto a gel forSDS-PAGE. After transfer onto a nitrocellulose membrane, immunodetectionwith the AB9040 antibody and the A-2066 antibody (equal loading control)is carried out. SS-x: normal breast biopsy number x, ST-x: tumoralbreast biopsy number x.

The results are given in FIG. 2, which represents a photograph of aWestern blot showing the presence of ProNGF in the cancerous mammarybiopsies (ST-1 to -4) but not in the normal biopsies (SS-1 to -4), actinhaving been used as positive control.

Detection Using Mammary Biopsies, by Immunohistochemistry

Tissue array slides were used to screen a large number of samples. Theseare mammary biopsies spotted onto slides. The tissue arrays used herecontain 60 spots of biopsies corresponding to one control, four normaldonors and 25 patients suffering from breast cancer, in duplicate. Thecharacteristics of the patients and also the intensity of theimmunolabeling with the anti-ProNGF antibody are given in table 1. Inthe mammary biopsies derived from normal donors, weak labeling ispresent (intensity + in 4/4 patients), whereas the signal is muchstronger in the cancerous mammary tissues (intensity ++/+++ in 20/25patients). Furthermore, the labeling profile is different between thetwo types of tissues: in the cancerous tissue, the labeling isessentially epithelial, whereas in the normal biopsies, it is not.

TABLE 1 Characteristics of mammary biopsies present on the breast cancertissue array and intensity of immunohistochemical labeling with theanti-ProNGF antibody. Label of TNM Identifier Diagnosis differentiationclassification Stage Labeling intensity HT00031 Control 0 HT00500 Normalbreast + HT00501 Normal breast + (no epithelium) HT00502 Normal breast +(no epithelium) HT00503 Normal breast + (no epithelium) HT00509 Invasiveducal carcinoma Medium T2N0M0 IIB 0 HT00510 Invasive ductal carcinomaMedium T2N0M0 IIB + + HT00511 Invasive lobular carcinoma High T2N11M0IIIB + + HT00512 Invasive lobular carcinoma High T2N17M1 IV + + +HT00513 Invasive lobular carcinoma Medium T2N1M0 IIB + HT00520 Invasiveductal carcinoma Medium T2N1M0 IIB 0 HT00521 Invasive ductal carcinomaMedium T2N0M0 IIB + + HT00522 Invasive ductal carcinoma Medium T2N1M1IV + + + HT00523 Invasive ductal carcinoma Medium T2N3M1 IV + + HT00524Invasive ductal carcinoma Medium T2N4M1 IV + HT00531 FibroadenomaUnknown T2N0M0 IIB 0 HT00532 Invasive ductal carcinoma Medium T2N2M0IIIA + + HT00533 Invasive ductal carcinoma Low T2N0M0 IIB + + + HT00534Invasive ductal carcinoma Low T2N1M0 IIB + + HT00535 Invasive ductalcarcinoma Medium-Low T2N1M0 IIB + + + HT00542 Invasive ductal carcinomaMedium T2N0M0 IIB 0 HT00543 Invasive ductal carcinoma Medium T2N0M0IIB + + + HT00544 Invasive ductal carcinoma Medium T2N0M0 IIB + + +HT00545 Invasive ductal carcinoma Medium T2N0M0 IIB + + + HT00546Invasive ductal carcinoma Medium T2N0M0 IIB + + + HT00553 Invasiveductal carcinoma Medium T2N3M0 IIIB 0 HT00554 Invasive ductal carcinomaLow T2N1M0 IIB + + + HT00555 Invasive ductal carcinoma Medium T2N0M0IIB + + + HT00556 Invasive ductal carcinoma Medium T2N0M0 IIB + + +HT00557 Invasive ductal carcinoma Medium T2N0M0 IIB + + +

Detection Using Lung and Thyroid Biopsies by Immunohistochemistry

Other tissue array slides were used to analyze the expression of ProNGFin cancer types other than breast cancer. We screened nine patients foreach cancer and analyzed, in parallel, the tumoral tissue and the normaltissue taken from the same patient in proximity to the tumor. Thus, wewere able to demonstrate an overexpression of ProNGF in the case of lungcancer and thyroid cancer. The characteristics of the patients and alsothe intensity of the immunolabeling with the anti-ProNGF antibody aregiven in table 2. For lung cancer as for thyroid cancer, the labeling ismuch stronger in the tumoral tissue than in the normal tissue in 7patients out of 9. For the other patients (2/9 for each cancer), thesignals keep the same intensity in the tumoral and normal tissues. Itshould be recalled that our analytical technique is onlysemiquantitative, and that it cannot reveal small differences inexpression.

TABLE 2 Characteristics of the lung and thyroid biopsies present on thetissue array and intensity of the immunohistochemical labeling with theanti-ProNGF antibody. Labeling intensity Level of TNM Cancerous NormalIdentifier Diagnosis differentiation classification Stage tissue tissueLung cancers P1 Adenocarcinoma High T2N0M0 IB + + + + + P2 Large cellcarcinoma T2N0M0 IB + + + + + P3 Epidermoid cancer Medium T2N0M0 IB+/+ + + + P4 Epidermoid cancer Medium T2N1MO IIB + + + + P5 Epidermoidcancer Medium T4N0M0 IIIB + + + P6 Adenocarcinoma High T2N0M0 IB + + +P7 Epidermoid cancer Medium T2N1M0 IIB + + + + P8 Epidermoid cancerMedium T3N2M0 IIIA + + + + P9 Epidermoid cancer High T2N1M0 IIB + + + +Thyroid cancers T1 Papillary carcinoma T2N0M0 I + + + T2 Papillarycarcinoma T2N0M0 I + + + + T3 Papillary carcinoma T2N0M0 I + + + + + +T4 Papillary carcinoma T2N0M0 II + + + +/+ + + +/+ + T5 Papillarycarcinoma T2N0M0 II + + T6 Papillary carcinoma T3N1aM0 III + + + + T7Papillary carcinoma T1N0M0 I + + + + + +/+ + T8 Papillary carcinomaT2N1bM0 IVA + + + +/+ + T9 Papillary carcinoma T3N0M0 III + +

Secretion of ProNGF

In order to determine whether the ProNGF is secreted, we concentratedMCF-7-conditioned medium. To do this, 100 μL of conditioned mediumconcentrated 224 times are subjected, after electrophoresis andtransfer, to immunodetection with the SC-548 antibody. Recombinant NGFand recombinant ProNGF were analyzed as controls.

The results are given in FIG. 3A, which represents a photograph of aWestern blot showing the secretion of ProNGF by the MCF-7 cancerouscells and the absence of secretion of NGF (concentrated conditionedmedium lane), the two lanes to the right being control lanes. It may benoted that no NGF secretion is observed here because the experiment isstopped too early to observe NGF due to the abundance of ProNGF. Whenthe experiment is continued, a band for NGF is also observed. Thisdemonstrates that, not only is ProNGF a tumor marker, but it is alsosecreted in large amounts.

To verify that the concentrating/desalifying unit used, with a cut-offthreshold of 10 kDa, was capable of retaining NGF (13.6 kDa), weconcentrated a mixture of recombinant ProNGF and recombinant NGF underthe same experimental conditions: 20 ng of ProNGF and 20 ng of NGF wereadded to unconditioned medium and were then subjected to the sameconditions as the conditioned medium.

The results (FIG. 3.B) prove that the concentrating/desalifying unit iscapable of retaining NGF and ProNGF.

Detection in the Serum

100 μg of protein extracts of mouse sera are loaded onto a gel forSDS-PAGE. After transfer onto a nitrocellulose membrane, immunodetectionwith the AB9040 antibody is carried out. In the equal loading control, aband visible at 24 kDa on the membrane, after staining with Ponceau red,is shown. Cancerous: 6 sera from mice injected with MDA-MB-231 cells,Normal: 2 sera from mice injected with PBS.

The results are given in FIG. 4, which shows that a band immunoreactivefor ProNGF appears at 26 kDa (weight of ProNGF) in 5 out of 6 sera frommice injected with cancerous cells; and no band was detected in the serafrom the control animals.

Detection by Mass Spectrometry

We carried out the procedure as indicated above. The results are givenin FIG. 5, which represents graphs of detection, by mass spectrometry,of ProNGF in the MCF-7-conditioned medium (FIGS. 5A, 5C and 5E) or ofrecombinant ProNGF (FIGS. 5B and 5D), giving the relative abundance as afunction of the mass. FIGS. 5A and B correspond to the protein fractionfrom the nanoLC corresponding to the SIM of at least one of the threecharacteristic ions of ProNGF. FIG. 5C corresponds to the mass spectrumof the protein eluted at 29.43 min in A. FIG. 5D corresponds to the massspectrum of recombinant ProNGF with its three characteristics ions inred. Finally, FIG. 5E shows the deconvolution of the mass spectrum ofthe protein eluted at 29.43 min in A.

These results show that a protein corresponding to the SIM (Selected IonMonitoring) of ProNGF, which is eluted at the same time (29.43 min, FIG.5.A) as recombinant ProNGF (30.17 min, FIG. 5.B), appears in theMCF-7-conditioned medium. This protein, once ionized, generates the samemulticharged ions (998.2, 1039.9, 1083.4, FIG. 5.C) as the recombinantProNGF (996.2, 1037.5, 1082.6, FIG. 5.D). Furthermore, the calculationby deconvolution of the mass of the protein gives a value of 25 971 Da(FIG. 5.E). Thus, these results show that, firstly, we indeed identifiedProNGF in the MCF-7-conditioned medium, and that, secondly, thedetection of ProNGF by mass spectrometry is possible.

EXAMPLE 3 ProNGF Therapeutic Target

3.1. Methodology

Expression of Sortilin ProNGF Receptor

The methods for carrying out the protein extraction are identical tothose described for ProNGF (see point 2.1 above).

For the Western blotting, the membranes are saturated with 4% bovineserum albumin in a solution of TBS (Tris Buffered Saline) 0.1% Tween 20(17.54 g NaCl, 2.42 g Tris, 2 mL tween 20, QS 2 L, pH adjusted to 7.4)at ambient temperature for 2 hours. After saturation, the membranes areincubated overnight at 4° C. with the various primary antibodies:anti-Sortilin (BD612101 from BD Biosciences) at 1:2000, anti-Actin(A-2066 from Sigma) at 1:5000 in the saturation solution. After rinsingwith TBS 0.1% Tween 20, the membranes are incubated for 1 hour atambient temperature with an anti-rabbit secondary antibody (A-1949 fromSigma, France) coupled to horseradish peroxidase, at 1:20000 in thesaturation solution. After rinsing, visualization is carried out withthe ECL system (Pierce Interchim, France) according to the recommendeddata for use.

Transfection of Interfering RNA (siRNA)

The interfering RNA (siRNA) transfection experiments were carried outwith the Cell Line Nucleofector kit from Amaxa and the correspondingelectroporation apparatus. This system makes it possible to obtain ahigh efficiency of gene transfer to the nucleus (nucleofection).

The MDA-MB-231 cells are passaged 2 days before the transfection, so asto reach 80% confluence at the time of the experiment. The cells to betransfected are trypsinized and then counted. The cell pellet (1 millioncells) is taken up in 100 μl of Nucleofector Kit V solution and 3 μg ofsiRNA are added. This cell suspension is transferred into anelectroporation cuvette, placed in the Amaxa apparatus andelectroporated according to the X-13 program. The cells are subsequentlyput back in culture in a well of a 6-well plate for 24-72 h. The cellproliferation is followed over time and the cells are counted at regulartime intervals. Anti-Sortilin Western blotting is carried out on thelysates recovered at various times, according to the protocol describedin the previous paragraph, “Expression of Sortilin”.

The MCF-7 cells are passaged 3-4 days before transfection, so as toreach 50% confluence at the time of the experiment. Two million cellsare taken up in 100 μl of Nucleofector Kit V solution and 3 μg of siRNAare added. The transfection program used on the Nucleofector is E-14.Anti-ProNGF Western blotting is carried out on the lysates recoveredafter 24 hours or after 48 hours, according to the protocol described inpoint 2.1. The relative amount of ProNGF detected by blotting isevaluated with the QuantityOne software (Bio-Rad), related to the equalloading (actin) and presented in the form of a histogram where 100% isassigned to the siGFP control condition.

ProNGF Biological Activity Measurements

A “starving” medium was used, “starving” denoting a medium identical tothe complete medium but without serum and supplemented with 2 μg/mL offibronectin and 30 μg/mL of transferrin. For the various biologicalactivity measurements, the various test molecules (ProNGF, NGF andgalardin) are added to this starving medium. During the treatments, themedia are renewed every 24 hours.

Migration and Invasion

The migration and invasion tests are carried out as previously describedby Bracke et al. (1999, J Natl Cancer Inst, 91: 354-359). The protocolsfor using Boyden chambers and for invasion in collagen gel are describedbelow.

Boyden Chamber (Transwell®)

12-well plates containing Transwells® with a pore diameter of 12 μm areequilibrated with starving medium and placed at 37° C. under 5% CO₂ for2 hours. The medium is then drawn off and replaced, in the lowerchamber, by starving medium with the various test molecules, whereas, inthe upper chamber, 40 000 cells are seeded in starving medium alone.After 24 hours, the Transwells® are rinsed with PBS, the upper face isscraped, and then Hoechst labeling is carried out (as described for thesurvival test), so as to visualize the cells that have crossed themembrane. The Transwells® are mounted between a slide and cover slip bymeans of a drop of glycergel heated to 55° C., and the slides are thenconserved at 4° C. in the dark until counting. Each condition is carriedout in duplicate; a minimum of 40 fields is counted for each condition.The data represent the mean, weighted by the standard deviations, of thecounts on these 40 fields. They are provided as percentage migrationwhere 100% migration is assigned to an HGF control at 50 ng/mL.

Collagen Gel Invasion Test

The collagen type I gel is prepared in the following way: 2.1 mL ofcollagen Type I, 0.8 mL of EMEM (10×), 4.6 mL of PBS, 0.8 mL of NaHCO₃at 0.25 M and 0.15 mL of 1 M NaOH. 1.25 mL are deposited in the wells ofa 6-well plate. Once the gel has solidified, 100 000 cells are seededwith starving medium and the various test molecules for 24 hours. Thecells are subsequently counted and the invasion index is determined(number of deep cells related to the number of cells at the surface).Each condition is carried out in triplicate and a minimum of 45 fieldsis counted per condition.

3.2. Results

Expression of Sortilin, ProNGF Receptor

50 μg of total protein extracts of mammary epithelial cells are loadedonto a gel for SDS-PAGE. After transfer onto a nitrocellulose membrane,immunodetection with the BD612101 antibody and the A-2066 antibody(equal loading control) is carried out. NMEC: normal mammary epithelialcells, BT-20, MCF-7, MDA-MB-231 and T-47D are cancerous mammaryepithelial cell lines.

The results are given in FIG. 6, which represents a photograph of aWestern blot showing the presence of Sortilin in the cancerous mammaryepithelial cells (MCF-7, T47-D, BT-20 and MDA-MB-231) and the normalcells (NMEC cells), actin having been used as positive control. Sortilintherefore appears in all these cells. On the other hand, this bandappears to be more intense in the cancerous cells than in the normalcells, whereas the loading control, actin, does not vary. One maytherefore think that there is less Sortilin in the normal mammaryepithelial cells than in the cancerous cells.

Decrease in the Expression of Sortilin by Interfering RNA (SortilinKnock Down)

MDA-MB-231 cells maintained in the EMEM culture medium were transfectedeither with culture medium alone (Mock), or with an interfering RNAdirected against the GFP protein (siGFP) or with an interfering RNAdirected against Sortilin (siSORT).

The results given in FIG. 7 show that, under the culture conditionschosen, the number of MDA-MB-231 cells in culture does not increase overtime in the presence of siSORT. In the Mock or siGFP control groups, thenumber of cells doubles between 24 and 48 h of culture. Theproliferation of the MDA-MB-231 cancerous mammary line is thereforeslowed down by transfection of an interfering RNA directed againstSortilin. We verified by Western blotting that the siSORT transfectionindeed decreased the level of expression of the Sortilin protein. Thus,this experiment allowed us to show that it was possible to control theabnormal proliferation of the breast cancer cells by targeting Sortilin,the ProNGF receptor, with an interfering RNA.

Migration

The effects of ProNGF on the migration of mammary epithelial cells werecomprehended by means of tests in Boyden chambers (Transwell®). Eachcondition was carried out in duplicate and a minimum of 40 fields wascounted. The MCF-7 cells were seeded in starving medium at the upperface of the Transwell®, whereas the lower face bathed in starving mediumalone or with 200 ng/mL of ProNGF, or with 20 μM of galardin with orwithout 200 ng/mL of ProNGF, or with 200 ng/mL of NGF. The cells havingcrossed the Transwell® were then counted. *p<0.05, comparison with thestarving medium alone condition.

The results are given in FIG. 8, which represents a graph giving thepercentage cell migration using MCF-7 cells bathed in the various media.These results demonstrate that treatment of the MCF-7 cancerous cellswith ProNGF makes it possible to increase their ability to migrate, in asimilar manner to NGF at equal doses. The addition of galardin, which iscapable of inhibiting the synthesis of NGF from ProNGF, to the culturemedium tends to demonstrate that the promotion of the migratory activityis due to the ProNGF and not to the NGF that it is capable ofgenerating.

Invasion

The invasive capacity of the ProNGF-stimulated MCF-7 cells was tested incollagen type I gel. Each condition is carried out in triplicate and aminimum of 45 fields is counted per condition. The MCF-7 cells areseeded onto a collagen type I gel in starving medium alone or with 200ng/mL of ProNGF, or with 20 μM of galardin with or without 200 ng/mL ofProNGF, or with 200 ng/mL of NGF. *p<0.05, comparison with the starvingmedium alone condition.

The results are given in FIG. 9, which represents a graph showing theinvasion index using MCF-7 cells bathed in the various media. Thesetests show that ProNGF stimulates MCF-7 invasion.

Decrease in the Expression of ProNGF by Interfering RNA

The MCF-7 cells were transfected with 3 μg interfering RNA (siRNA)directed against GFP (siGFP) or against ProNGF (siProNGF). After 24 to48 h of culture, the cells were lysed and ProNGF Western blotting wascarried out. The relative amount of ProNGF detected by blotting wasevaluated with the Quantityone software (Bio-Rad) related to the actinequal loading and presented in the form of a histogram where 100% isassigned to the siGFP control condition (FIG. 10). On the photographs ofthe blots, we were already able to observe that the transfection ofsiProNGF decreases the expression of the ProNGF protein in the MCF-7cells. Densitometric analysis made it possible to evaluate this decreaseat 59% after 24 h of transfection.

This experiment allowed us to show that it was possible to decrease thelevel of expression of ProNGF in breast cancer cells by using theinterfering RNA strategy.

1. A method for in vitro diagnosis of cancer, comprising determining thepresence of ProNGF in a biological sample derived from a patientsuspected of suffering from cancer.
 2. The method as claimed in claim 1,wherein the cancer is breast, thyroid or lung cancer.
 3. The method asclaimed in claim 2, wherein the cancer is breast cancer.
 4. The methodas claimed in claim 2, wherein the cancer is lung cancer.
 5. The methodas claimed in claim 2, wherein the cancer is thyroid cancer.
 6. Themethod as claimed in claim 1, wherein the presence of ProNGF isdemonstrated by direct detection of ProNGF in said biological sample. 7.The method as claimed in claim 6, wherein the detection of ProNGF iscarried out by means of an immunoassay or by mass spectrometry.
 8. Themethod as claimed in claim 6, wherein said biological sample consists ofbiological fluid or of a tissue originating from a biopsy of a tumor ormetastases.
 9. The method as claimed in claim 8, wherein the biologicalsample consists of a tissue originating from the biopsy of the tumor orthe metastases of the patient.
 10. The method as claimed in claim 8,wherein the biological sample consists of biological fluid, pretreatedin order to isolate circulating tumor cells contained in said fluid. 11.The method as claimed in claim 10, wherein the circulating tumor cellsare subsequently cultured under conditions such that they secreteProNGF.
 12. The method as claimed in claim 10, wherein the circulatingtumor cells are cultured under conditions which make it possible toblock the ProNGF inside said cells.
 13. The method as claimed in claim1, wherein the detection of ProNGF is carried out by culturingProNGF-sensitive cells in the presence of said biological sample. 14.The method as claimed in claim 13, wherein said biological sampleconsists of a biological fluid sample, pretreated in order to isolatecirculating tumor cells contained in said fluid.
 15. The method asclaimed in claim 14, wherein the circulating tumor cells aresubsequently cultured under conditions such that they secrete ProNGF.16. A method for the early diagnosis, screening, therapeutic follow-up,prognosis or diagnosing relapses in cancer, comprising the in vitrodiagnosis of cancer according to claim
 1. 17. A method for treatment ofa patient suffering from cancer, comprising administering atherapeutically effective amount of a ProNGF inhibitor to said patient.18. The method as claimed in claim 17, wherein the amount is effectivefor blocking cell migration or invasion in patients suffering fromcancer.
 19. The method as claimed in claim 17, wherein the ProNGFinhibitor is an anti-ProNGF antibody or a ProNGF receptor analog insoluble form.
 20. The method as claimed in claim 17, wherein said ProNGFinhibitor has been previously placed under conditions such that itspecifically penetrates into cells of interest.
 21. The method asclaimed in claim 17, wherein the ProNGF inhibitor is an siRNA of aProNGF receptor.
 22. A pharmaceutical composition, comprising as activeingredient, at least one ProNGF inhibitor.
 23. The method as claims inclaim 10, wherein the biological sample is pretreated in order toisolate circulating tumor cells contained in said fluid.
 24. The methodas claims in claim 14, wherein the biological sample is pretreated inorder to isolate circulating tumor cells contained in said fluid.